We have devised a very simple procedure that allows us to attach a wide variety of organic molecules to the terminal phosphate of an unprotected oligonucleotide or polynucleotide. During the next grant period we plan to exploit this chemistry to develop a number of techniques that have broad general application. 1. We will attach a variety of porphyrins to the 5' terminus of an oligonucleotide to bring about the hybridization- dependent, specific and efficient cleavage of complementary DNA or RNA. Reagents of this kind would have many applications. They could, for example, be used as 'restriction enzymes' for single strand RNA (or DNA) or as probes to explore the tertiary structure of RNA molecules. 2. We will develop methods for the attachment of molecules that combine irreversibly with proteins to the 3'-terminus of oligonucleotides. We anticipate that, if we hybridize these reagents to a messenger RNA, the reactive groups will attack a protein at the messenger-entry site of the ribosome during protein synthesis. We hope to learn how to inhibit intracellular protein synthesis in a highly specific manner by inactivating ribosomes. Only the ribosomes in cells containing an RNA complementary to an oligomer should be affected by a protein-modifying adduct of that oligomer. In the long run, when adequate in vivo delivery methods become available, adducts of this type might find many applications in medicine. 3. We will develop a hybridization assay for DNA that makes use of short RNA substrates that are replicated exponentially by QB RNA polymerase as reporters. We have prepared an adduct in which QB-Midivariant I RNA is joined to avidin by a cleavable linker. This adduct replicates normally, with a doubling time of 34 seconds, thus producing about 10-12 descendents in 25 minutes. By joining this adduct to an oligodeoxynucleotide, we plan to develop a non-radioactive probe assay at least 100 times more sensitive than the best presently available non-radioactive assays. A sensitive assay of this kind would have many important applications in diagnostic medicine.